The major objectives of this proposal are: (1) to investigate structure-function relationships and biosynthesis of tRNAs and (2) to extend our studies on site-specific mutagenesis of tRNA genes to generate a variety of suppressors which function in mammalian cells. Our approach is to alter single nucleotides at predetermined sites in tRNA genes, introduce them into the appropriate organism and examine the effect of these alterations on the function of the tRNA in vivo. Both prokaryotic and eukaryotic tRNAs will be studied. Should the tRNA genes be defective in function in vivo we shall determine whether their biosynthesis is impaired. If biosynthesis is unaffected, we shall purify the mutant tRNAs and by in vitro studies identify the step at which they are defective in function (aminoacylation, binding to elongation factors, ribosome etc.). We shall use this approach to study (a) the role of the invariant nucleotides in tRNA biosynthesis and in function, and (b) the relationship between the unique structural features present in initiator tRNAs and their special properties. The role of the highly conserved -TPsiC- sequence in tRNA function will also be determined. Finally, we propose to extend our recent work on generation and cloning of an amber suppressor tRNA gene in mammalian cells to include a variety of nonsense suppressors which will function in mammalian cells. The eventual goal is to establish stable cell lines which contain different suppressor tRNA genes integrated into their cellular DNA. In a continuation of our studies of tRNAs and tRNA genes in N.crassa mitochondria, we propose to study enzymes involved in their biosynthesis. We shall determine whether the highly conserved palindromic DNA sequences which flank tRNA genes in N.crassa mitochondria play a role in RNA processing. In addition, we shall purify the tRNA processing enzyme "RNase P" from N.crassa mitochondria and determine whether it has an essential RNA component like the E.coli enzyme and, if so, whether this RNA is mitochondrially coded.